Bioorganic & Medicinal Chemistry Letters 20 (2010) 3036–3038
`
`Contents lists available at ScienceDirect
`
`Bioorganic & Medicinal Chemistry Letters
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`j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b m c l
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`A convenient synthesis of (Z)-4-hydroxy-N-desmethyltamoxifen (endoxifen)
`
`Abdul H. Fauq *, Ghulam M. Maharvi, Dola Sinha
`
`Chemical Synthesis Core Facility, Mayo Clinic Jacksonville, FL 32246, USA
`
`a r t i c l e
`
`i n f o
`
`a b s t r a c t
`
`Article history:
`Received 17 February 2010
`Revised 29 March 2010
`Accepted 31 March 2010
`Available online 3 April 2010
`
`Keywords:
`LY411575
`Endoxifen
`Tamoxifen
`Semipreparative HPLC
`
`A mixture of the (Z)- and (E)-isomers of 4-hydroxy-N-desmethyltamoxifen was conveniently prepared in
`four steps. These geometrical isomers were then neatly separated by semi-preparative Reverse Phase
`High Performance Liquid Chromatography (RP-HPLC) using specified conditions. Additionally, the iso-
`lated E-isomer could be equilibrated in aqueous strong acid in acetonitrile or trifluoroacetic acid/dichlo-
`romethane to give a clean 1:1 mixture of Z/E isomers that was re-subjected to HPLC separation. In this
`way, most of the undesired (E)-isomer could be readily converted to the desired (Z)-isomer providing
`quick access to over 200 mg quantities of pure endoxifen (Z-isomer), a potent antiestrogenic metabolite
`of tamoxifen traditionally used in breast cancer treatment.
`
`Ó 2010 Elsevier Ltd. All rights reserved.
`
`1-[4-(2-Dimethylaminoethoxy)-phenyl]-1,2-diphenylbut-1(Z)-
`ene (tamoxifen (TAM)) is a non-steroidal antiestrogen drug widely
`used for breast cancer treatment.1 The pharmacological profiles of
`tamoxifen indicate that it elicits its anti-cancer activity through its
`active metabolites 4-hydroxytamoxifen (4-OH-TAM) and its desm-
`ethyl analogue endoxifen that are generated by the action of hepatic
`CYP 2D6 and 3A4 isozymes on tamoxifen after hydroxylation fol-
`lowed by N-demethylation.2,3 It is established that some patients
`do not derive therapeutic benefit from the administration of tamox-
`ifen or even suffer relapses because of their inherent genotypic con-
`straints.3 Co-administration of certain drugs (e.g., paroxetine or
`fluoxetine) also have demonstrated interactive inhibitory effect on
`CYP 2D6 and other cytochrome P450 enzymes. This of lack of activity
`results from reduced availability of therapeutic levels of the active
`metabolite (Z)-desmethyl-4-hydroxytamoxifen (endoxifen).4 Re-
`cently, Hawse and co-workers have shown that endoxifen is the ac-
`tual anti-estrogenic drug that works by degrading the estrogen
`receptor and not by its inhibition.3 Therefore, in order to facilitate
`human tissue studies as well to explore the possibility that it may
`be appropriate in special cases to consider a dose regimen that, in-
`stead of tamoxifen, includes the active metabolite endoxifen, rela-
`tively large quantities of endoxifen may be required. A search of
`the literature showed that while low level milligram quantities of
`the endoxifen have been purified by analytical HPLC,5 synthesis
`and purification of relatively large quantities of endoxifen have not
`been reported. Herein, we report synthesis and purification proto-
`cols that have resulted in production of pure endoxifen in excess of
`200 mg quantities for animal studies and tissue work in less than
`
`* Corresponding author. Tel.: +1 904 953 8034; fax: +1 904 953 7117.
`E-mail address: fauq.abdul@mayo.edu (A.H. Fauq).
`
`0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
`doi:10.1016/j.bmcl.2010.03.117
`
`two weeks time. This work involved a short four-step synthesis of
`a mixture of endoxifen and the (E)-isomer and the use of semi-pre-
`parative reverse phase HPLC columns for their separation. Neverthe-
`less, much larger quantities of endoxifen are expected to be
`conveniently generated by employing preparative columns.
`Apart from a stereoselective synthesis of tamoxifen involving car-
`bometalation of alkynylsilanes,6 earlier reported syntheses of (Z)-4-
`hydroxytamoxifen, a precursor of endoxifen, were somewhat
`cumbersome and non-stereoselective.7 A ground-breaking stereose-
`lective synthesis of (Z)-4-hydroxytamoxifen by Gauthier and Labrie
`involved McMurry reaction as a key step.8 Even though the synthesis
`of endoxifen was not reported, the authors managed to obtain a favor-
`able 14:1 ratio of the (E)- and (Z)-isomers of 1-(4-hydroxyphenyl)-1-
`[4-(trimethylacetoxy)phenyl]-2-phenylbut-1-ene (3b:4a,Fig. 1) after
`reacting the monopivaloyl derivative of 4,40-dihydroxybenzophe-
`none (1b) with propiophenone in the McMurry reaction simply by
`manipulating proportions of TiCl4 and Zn. The ratio 14:1 was en-
`hanced to 100:1 by trituration of the crude with methanol. In our
`hands, the crude obtained after the McMurry reaction was directly
`chromatographed on silica gel to obtain desired (E)-stereoisomer in
`88% yield. In this reaction, the phenolic and the ethyl components
`were shown to preferentially align trans to each other as prece-
`dented.9 We carried out the reported four-step Gauthier–Labrie syn-
`thetic sequence through the intermediate 5 to (Z)-4-OH-TAM 3c.
`Unfortunately, attempted demethylation of 5 or the (Z)-4-OH-TAM
`3c itself using drastic conditions (vinyl chloroformate in dioxane at
`135–170 °C in sealed tube) as reported5c or ethyl chloroformate5a,10a
`in refluxing toluene produced a significantly stereoscrambled mix-
`ture of endoxifen and its (E)-isomer in low yield. In using some other
`milder carbamate-mediated N-demethylation conditions,10b–d it was
`noticed that lower yields and significant stereorandomization of the
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`A. H. Fauq et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3036–3038
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`3037
`
`O
`
`RO
`
`OH
`
`1a, R = H
`1b, R = pivaloyl
`2, R = CH2CH2N(Me)2
`
`RO
`
`OR'
`
`RO
`
`OR'
`
`3a, R = H; R' = H
`3b, R = pivaloyl; R' = H
`3c, R = H; R' = -CH2CH2N(Me)2
`((Z)-4-OH-TAM)
`5, R = pivaloyl; R' = -CH2CH2N(Me)2
`6a, R = pivaloyl;
`R' =CH2CH2N(CH3)C(O)OCH(Cl)CH3
`6b, R = pivaloyl;
`R' = CH2CH2N(CH3)C(O)OCH2CH3
`8a, R = H; R' = CH2CH2NHCH3 (endoxifen)
`
`4a, R = pivaloyl; R' = H
`4b, R = H; R' = CH2CH2N(Me)2
`((E)-4-OH-TAM)
`7a, R =CH2CH2N(CH3)C(O)OCH(Cl)CH3;
`R' = pivaloyl
`7b, R = pivaloyl;
`R' =CH2CH2N(CH3)C(O)OCH2CH3
`8b, R = H;
`R' = CH2CH2NHCH3 (E)-isomer
`
`Figure 1. Endoxifen and reported intermediates for its synthesis.
`
`double bond could not be avoided. Of the various carbamate-medi-
`ated demethylation protocols, the best reaction conditions entailed
`heating a mixture of 2-chloroethyl chloroformate and (Z)-4-OH-
`TAM 3c in dichloroethane which resulted in stereorandomization of
`the tetrasubstituted double bond to the extent of 20% furnishing a
`4:1 mixture of the desired endoxifen carbamoyl precursor (6a) and
`its (Z)-isomer (7a) in 79% yield (Fig. 1). The mechanism of the isomer-
`ization during carbamate-mediated N-demethylation is currently
`speculative, albeit precedented.5a Since the components of this ste-
`reoisomeric mixture could not be separated, it was directly reacted
`with methyllithium at 78 °C in tetrahydrofuran which concomi-
`tantly cleaved carbamoyl and pivaloyl ester moieties to generate a
`4:1 mixture of endoxifen (8a) and its (E)-isomer (8b). The overall
`six-step synthetic sequence furnished a mixture of endoxifen 8a
`and its (E)-isomer 8b in 26% combined yield.
`With the hope of retaining stereochemical integrity during
`demethylation, we attempted to synthesize pure carbamoyl (E)-
`isomer of 6b by reacting 3b (Scheme 1) either with the ethyl (2-
`hydroxyethyl)(methyl)carbamate (10, prepared by reacting the
`alcohol with ethylchloroformate/triethylamine in DCM;
`cf.
`Supplementary data) under Mitsunobu conditions, or by SN2
`reaction between 3b and 2-bromo or 2-iodo-derivative of ethyl
`(2-hydroxyethyl)(methyl)carbamate (prepared by reacting ethyl
`(2-hydroxyethyl)(methyl)carbamate 10 with CBr4, triphenylpho-
`shine/DCM to give 11a, or with I2/triphenylphoshine/imidazole/
`toluene to give 11b under basic (Cs2CO3/DMF); cf. Supplementary
`data). While the Mitsunobu reaction completely retained stereo-
`chemistry of the substituted product 6b, the basic conditions
`(Cs2CO3/DMF) employed during bromo- or iodo-displacement re-
`sulted in 30% stereoscrambling to give 7b. This unwelcome reac-
`
`tion outcome suggested that both acidic (e.g., treatment with
`DCM/TFA mixture, vide infra) as well as the basic milieu compro-
`mised the stereochemical integrity of the electron-rich tetrasubsti-
`tuted double bond. The labile nature of the stereochemistry under
`basic conditions was further evidenced when an attempt was
`made to achieve simultaneous removal of the pivaloyl and ethoxy-
`carbonyl moieties from pure 6b (derived from Mitsunobu reaction)
`with methyllithium under the usual mild (78 °C) conditions. This
`reaction also produced ca. 30% undesired (E)-isomer 8b. Similarly,
`when methyllithium was mixed with pure 8b in THF at 78 °C,
`30% of endoxifen 8a was obtained. While the acid-catalyzed stere-
`orandomization of the tetrasubstituted double bond is expected,
`the base-mediated partial isomerization during the ethoxycar-
`bonyl removal may be explained by invoking the possibility of res-
`onance in phenolate anion playing a part in generating a partial
`single bond character in the para-substituted sp2-hybridized ben-
`zylic carbon of the phenol moiety.
`Since it was important to rapidly produce over 200 mg of
`endoxifen for use in several ongoing anti-breast cancer investiga-
`tions, the problem of separating the final 4:1 endoxifen:(E)-isomer
`mixture presented a challenge. Even though RP-HPLC separation of
`endoxifen from its (E)-isomer has been achieved in small quanti-
`ties for in vitro studies,5c–e protocols for larger scale separation
`were not reported. Our attempts using the reported RP-HPLC con-
`ditions for larger scale separation using semi-preparative RP-HPLC
`columns and phosphate or triethylamine-containing buffers gave
`poor resolution of the endoxifen and its (E)-isomer. Additionally,
`silica gel chromatography using various eluents containing basic
`additives was unsuccessful. After considerable experimentation,
`success was finally realized when the RP-HPLC separation of the
`
`OH
`
`NH
`9
`
`EtOC(O)Cl/TEA/
`
`DCM
`
`OH
`
`N O
`O
`10
`
`CBr4/TPP/THF
`
`or, I2/TPP/imidazole
`
`Br
`
`N O
`O
`11a
`
`or
`
`I
`
`N O
`O
`11b
`
`OEt
`
`O
`
`ON
`
`7b
`
`+
`
`PivO
`
`OEt
`
`O
`
`ON
`
`10, TPP, DEAD,
`
`PivO
`
`OH
`
`or, 11a,/ 11b, Cs2CO3
`
`PivO
`
`3b
`
`6b
`
`Scheme 1.
`
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`3038
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`A. H. Fauq et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3036–3038
`
`.HCl
`
`NH
`
`O
`
`O
`
`9
`
`HO
`
`(i) CH3CH(Cl)OCOCl
`DCE, 0 ºC to reflux
`
`(ii) 6M HCl, MeOH
`reflux, 4 h
`
`2
`
`O
`
`NH
`
`O
`
`HO
`8b (E)-isomer)
`
`equilibration
`
`+
`
`.HCl
`
`NH
`
`Zn, TiCl3, THF
`reflux, 7h
`
`HO
`
`O
`8a (endoxifen)
`
`Scheme 2. Modified synthesis of endoxifen.
`
`stereoisomeric mixture was attempted with isocratic elution with
`a buffer containing 50% of 20 mM triethylammonium bicarbonate
`in acetonitrile at pH 8.8. This protocol separated the two peaks well
`apart even under significant column overloads (Vydac column, C-8,
`2.2  25 cm, FR 8 mL/min: RT for endoxifen, 53 min; for (E)-isomer
`81 min). The identities of the two geometrical isomers were con-
`firmed by peak matching with reported NMR data5a as well as by
`its expected antiestrogenic activity.3
`In spite of the fact that the six-step protocol for the synthesis of
`endoxifen can be achieved from the published synthetic proce-
`dures from its precursor 4-OH-TAM, the confounding problem of
`significant double bond isomerization during the demethylation
`still remained and necessitated HPLC purification. It was, therefore,
`deemed pragmatic to shorten the overall synthesis of endoxifen by
`doing away with protection/deprotection of the hydroxyl group of
`4,40-dihydroxybenzophenone (1) altogether. Our overall four-step
`strategy that continues to rely on the McMurry reaction8 is given
`in Scheme 2. The N,N-dimethylethyl derivative 4,40-dihydroxyben-
`zophenone (2), made from 4,40-dihydroxybenzophenone 1 in 46%
`yield was demethylated using 2-chloroethyl chloroformate-medi-
`ated demethylation methodology as described above in 83% overall
`yield. However, instead of decarbamoylation with methyllithium
`furnishing lower yield of the deprotected product, the intermediate
`2-chloroethyl carbamate was decomposed with 6 M-HCl in reflux-
`ing methanol to give the secondary amine hydrochloride salt (9) in
`higher yield (83%, Scheme 2). The hydrochloride salt 9 was sub-
`jected to McMurry reaction with propiophenone furnishing a 90%
`chromatographed combined yield of a mixture of the endoxifen
`8a and the (E)-isomer 8b in 1:3 ratio. Fortunately, this unfavorable
`ratio was readily and cleanly enhanced to 1:1 by heating the iso-
`meric mixture in 6 N-HCl in aqueous acetonitrile for 6 h or by,
`more simply, stirring the mixture with 1:1 DCM/TFA for 1 h. The
`(Z)-and (E)-isomers were then separated using the RP-HPLC condi-
`tions as outlined above. Additionally, the undesired (E)-isomer
`(8b)-containing fractions obtained from the HPLC runs were com-
`bined and re-equilibrated cleanly to 1:1 mixture of endoxifen and
`8b either by heating with equal volume of 6 N-HCl at 60 °C for 4–
`6 h, or, by evaporating to dryness and stirring with 1:1 DCM/TFA at
`rt. In both cases, the equilibrated mixture was directly subjected to
`HPLC purification resulting in enhanced overall yield of the endox-
`ifen. The remarkably large RT difference between the two isomers
`achieved under specified buffer conditions was critical to their suc-
`cessful larger scale separation because semipreparative RP-HPLC
`column could be safely overloaded. This protocol also enabled stor-
`age of large quantities of endoxifen as 1:1 Z/E mixture at 15 °C
`under dark for extended periods of time, pending fresh isolation
`of endoxifen as and when needed.
`In sum, a short four-step methodology for rapid generation of
`endoxifen/(E)-isomer mixture, obtained in 34% overall yield, was
`
`combined with a highly efficient RP-HPLC protocol for separation
`of endoxifen from the (E)-isomer. This methodology was used to
`conveniently and rapidly prepare over 200 mg quantities of pure
`endoxifen as and when needed for animal and tissue studies.
`
`Acknowledgment
`
`The internal financial support provided for this synthetic pro-
`ject by Mayo Foundation is gratefully acknowledged.
`
`Supplementary data
`
`Supplementary data (the spectroscopic and RP-HPLC chromato-
`graphic data as well as synthetic procedures for all the reported
`intermediates, and those of the final products) associated with this
`article can be found,
`in the online version, at doi:10.1016/
`j.bmcl.2010.03.117.
`
`References and notes
`
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`
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`(a) Grafe, I.; Schickaneder, H.; Jungblut, P. W.; Ahrens, K. H. EP 287690 A1
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`10. Some other N-demethylation protocols employing various chloroformates, see:
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